Enzymes have excellent catalytic functions with substrate specificity, reaction specificity, and stereospecificity. Stereospecificity of enzymes, with some exceptions, are nearly absolute.
Recent precise research has increased the importance of optically active substances for use in drugs, pesticides, feeds, and perfumes. Since optical isomers sometimes have quite different biological activities, techniques for specifically obtaining the isomer are important. For example, D(R)-form thalidomide has no teratogenic activity, but its L(S)-form shows strong teratogenicity. The practical use of thalidomide racemate caused the drug injury incidents by thalidomide. Furthermore, if one enantiomer shows an effective biological activity, the other enantiomer may sometimes have no activity, rather, it may competitively inhibit the activity of the effective enantiomer because of the coexistence of both enantiomers. As a result, the biological activity of the racemate is reduced to half or less of the activity of the effective enantiomer. Accordingly, it is industrially important to obtain (synthesize or optically resolve) optically pure enantiomers.
For this objective, an effective procedure has been used widely to optically resolve racemates synthesized. However, an unnecessary enantiomer is always produced as a by-product with the procedure of resolution after synthesis; a problem has been left unsolved in economizing on raw material. Even if the recovered by-product is reused as a raw material, a definite amount of the by-product is always produced. Therefore, enzymatic optical resolution has drawn attention because it does not produce by-products and a bulk of liquid waste. Enzymatic optical resolution is a method of specifically producing a desired enantiomer by utilizing enzyme specificity. Since unnecessary enantiomers are barely synthesized by this method, it is easy to obtain products of high optical purity. In addition, this method is also advantageous in economizing on raw material.
Generally, L-amino acids are widely and largely utilized in seasonings, food and feed additives, and infusions, and are thus very highly demanded. L-amino acids have been produced mainly by direct fermentation using microorganisms. In addition, optical resolution in which N-acyl-DL-amino acids are hydrolyzed with L-aminoacylases is also a known method for producing L-amino acids. It has been utilized to industrially produce L-amino acids that are difficult to produce by fermentation.
L-aminoacylases are widely found in animals, plants, and microorganisms. They have been purified from various organisms, and their properties have been clarified. N-terminal amino acids of many proteins are considered to be N-acetylated in vivo. L-aminoacylases presumably regenerate N-acetyl-amino acids produced by decomposition of proteins to amino acids. Among L-aminoacylases, acylase that acts on N-acyl-L-glutamic acid is reported to be involved in arginine biosynthesis (Fruth et al., J. Gen. Microb, 125:1, 1981).
In contrast, D-amino acids have not been a subject of interest for a long time because they are nonprotein amino acids. D-amino acids were known to naturally occur only in small cyclic peptides, peptidoglycan of bacterial cell walls, and peptide antibiotics. However, D-amino acids have been demonstrated to be constituents of neuro peptides and to exist as binding forms in tooth enamel, the lens, and cerebral proteins, resulting in investigation of physiological significance and enzymatic synthesis of D-amino acids.
At present, DL-amino acids have been optically resolved by physicochemical, chemical, or enzymatic methods. The enzymatic methods are the most convenient and industrially applicable for, for example, continuously producing L-methionine from N-acetyl-DL-methionine using a bioreactor on which L-aminoacylase is immobilized. D-amino acids may also be produced using hydantoinase. The method consists of two-step enzymatic reactions. The first reaction uses D-specific hydantoinase to convert D,L-5-substituted-hydantoin, which is synthesized at low cost from aldehyde analogues, to a D-carbamyl derivative. The second reaction uses D-amino acid carbamylase.
Another method is known in which D-aminoacylase hydrolyzes N-acetyl-DL-amino acids to produce D-amino acids (Sugie et al., Argric. Biol. Chem., 44:1089, 1980; Tsai et al., J. Enzyme Microb. Technol., 14:384, 1992).
D-tryptophan is one of important D-amino acids used as a medicinal raw material and the like. D-tryptophan can be obtained by deacetylating N-acetyl-DL-tryptophan. However, D-aminoacylase capable of-efficiently catalyzing this reaction in a stereospecific manner is not yet known.
D-aminoacylase was first reported to be found in Pseudomonas sp. KT83 isolated from soil by Kameda et al. in 1952 (Kameda et al., Nature, 170:888, 1952). This enzyme hydrolyzed N-benzoyl derivatives of D-phenylalanine, D-tyrosine, and D-alanine. Thereafter, D-aminoacylases were derived from microorganisms below.
Genus Pseudomonas 
(Kubo et al., J. Antibiot., 43:550, 1980; Kubo et l., J. Antibiot. 43:556, 1980; Kameda et al., Chem. Pharm. Bull., 26:2698, 1978; Kubo et al., J. Antibiot. 43:543, 1980)
Genus Streptomyces 
(Sugie et al., Argric. Biol. Chem., 42:107, 1978; Sugie et al., Argric. Biol. Chem., 44:1089, 1980)
Genus Alcaligenes 
(Tsai et al., Appl. Environ. Microbiol., 54:984, 1988; Yang et al., Biosci. Biotech. Biochem., 56:392, 1992; Yang et al., Appl. Environ. Microbiol., 57:2767, 1991; Tsai et al., Microb. Technol., 14:384, 1992; Moriguchi et al., Appl. Environ. Microbiol., 54:2767, 1988; Sakai et al., FEBS, 289:44, 1991; Sakai et al., J. Ferment. Bioeng., 71:79, 1991; Sakai et al., Appl. Environ. Microbiol., 57:2540, 1991; Moriguchi et al., Biosci. Biotech. Biochem., 57:1145, 1993; Kayama et al., J. Biochem., 118:204, 1995; Moriguchi et al., Biosci. Biotech. Biochem., 57:1149, 1993)
Tsai et al. and Moriguchi et al. also clarified the characteristics of D-aminoacylase derived from microorganisms belonging to the genera Alcaligenes and Pseudomonas and the amino acid and nucleotide sequences of the enzymes. Moriguchi et al. found, by using different inducers, three types of D-aminoacylases derived from microorganisms belonging to the genera Alcaligenes and Pseudomonas (Wakayama et al., Biosci. Biotech. Biochem., 59:2115, 1995).
Furthermore, Moriguchi et al. determined the nucleotide sequences of these D-aminoacylases derived from a microorganism belonging to the genus Alcaligenes and compared them with L-aminoacylases derived from Bacillus stereothermophilus, human, and pig. The result demonstrated that these D-aminoacylases have a low homology with L-aminoacylases (Wakayama et al., Biosci. Biotech. Biochem., 59:2115, 1995).
As to Actinomycetes, Sugie et al. reported D-aminoacylase of a microorganism belonging to the genus Streptomyces (Sugie et al., Argric. Biol. Chem., 44:1089, 1980). However, the enzyme has not been purified yet, and its characteristics remain unknown.
Any of these known D-aminoacylases exhibit only low activities for N-acetyl-D-tryptophan and cannot be used for synthesizing D-tryptophan. With these points described above as background, it has been demanded to isolate D-aminoacylase capable of producing D-tryptophan stereospecifically using N-acetyl-DL-tryptophan as a substrate as well as a gene encoding the enzyme.